Cuantificación del virus de hepatitis B por la técnica PCR tiempo real / Quantification of viral hepatitis type B using the real-time PCR technique

La técnica de reacción en cadena de la polimerasa (PCR) ha determinado, en la cuantificación de virus, un avance especial para el manejo de infecciones crónicas por virus, en especial, para HIV, virus de hepatitis B y C. La cuantificación en tiempo real se realiza en el ABI PRISM Sequence Detection System. Se amplifica, específicamente, el fragmento pb del genoma del virus de hepatitis B. Se recomienda el HBV PCR kit para la toma de la muestra., determinándose un protocolo para el almacenamiento de la misma. Es importante saber que el congelar las muestras o el almacenamiento prolongado disminuyen la sensibilidad del método. Se puede almacenar por años si es a una temperatura de - 70º C. Tubos con heparina o pacientes heparinizado alteraran la determinación de la muestra. El limite inferior detectable del virus B es de 3.78 UI/ml y el mayor es 1.4 x 10¹¹ UI/ml, se determinan todos los genotipos desde A-H. Los pacientes HBeAg positivos, usualmente, tienen valores mayores a 1 x 10(6) UI/ml y los HBe negativos portadores inactivos por lo general, valores menores a 1 x 10(4).

The polymerase chain reaction technique (PCR) has determined a special advance in the virus quantification for the management of chronic viral infections, especially for HIV hepatitis virus type B and C. The real-time quantification is performed in the ABI PRISM Sequence Detection System. The fragment pb of the genome of the hepatitis type B virus is amplified. The HBV PCR kit is recommended for taking a sample and a protocol for storing it is determined. It is important to know that freezing the samples or keeping them for a long time decreases the sensitivity of the method. It may be stored for years if kept at a temperature of -70° C. Heparinized patients or tubes with heparin will alter the determination of the sample. The lower limit of detection of B virus is 3.78 Ul/ml and the higher limit is 1.4 x 10(11) Ul/ml. All genotypes from A-H are determined. The patients with HbeAg positive usually present higher values than 1 x 10(6) Ul/ml and the HBe negative inactive carriers usually get lower values than 1 x 10(4).

Comparison between Two Sets of Primer for HBV-DNA by Polymerase Chain Reaction (PCR) / 대한임상병리학회지

BACKGROUND: The HBV-PCR assay seems to be potentially valuable diagnostic tool for the evaluation of variable serologic status. However, the selection of the primer for HBV-PCR test may be very important because they can influence the HBV-PCR positivity. METHODS: We compared the results of primer HBV1/2 including famous 1896 and 1899 mutation sites with those of primer PHBV1/2 at precore/core region. HBV-PCR was tested in 87 HBsAg-positive patients using two sets of primers. The results were evaluated according to the primers and also compared the results with the clinical diagnosis and the alanine aminotransferase (ALT) level. RESULTS: The positive rate of PHBV primer was higher than HBV primer including mutation sites (nucleotide 1896 and 1899) in HBeAg-negative patients. According to the clinical diagnosis, the sensitivity of PHBV primer was higher than that of HBV primer in chronic hepatitis patients. There was no significant correlation between ALT level and HBV-PCR results. CONCLUSIONS: It is important that the selection of primer in HBV-PCR is important, because the primer including mutation sites may result in false negative results. PHBV primer used in this study could be useful for the detection of HBV-DNA by HBV-PCR.